CN114306341B

CN114306341B - Use of phthalazine derivatives for the treatment of liver fibrosis diseases

Info

Publication number: CN114306341B
Application number: CN202210197109.3A
Authority: CN
Inventors: 徐强; 徐忆竹; 吴兴新; 黄磊; 翟冰新; 朱启华
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2022-03-02
Filing date: 2022-03-02
Publication date: 2022-05-24
Anticipated expiration: 2042-03-02
Also published as: CN114306341A

Abstract

The invention discloses application of a phthalazine derivative in treating hepatic fibrosis diseases, relates to the field of biological medicine, and particularly relates to application of a phthalazine derivative shown in a compound (I) in treating hepatic fibrosis diseases. Pharmacodynamic tests prove that the compound (I) can inhibit hepatic stellate cell activation caused by TGF-beta and has obvious improvement effect on hepatic fibrosis of a carbon tetrachloride molding mouse, so that the compound (I) has the inhibition effect on the hepatic fibrosis and can be used for treating the hepatic fibrosis, and the compound (I) can be used for preparing a medicament for treating the hepatic fibrosis diseases.

（I）。

Description

Use of phthalazine derivatives for the treatment of liver fibrosis diseases

Technical Field

The invention relates to the field of medicines, in particular to application of phthalazine derivatives in treating hepatic fibrosis diseases.

Background

Chinese patent CN201810140689.6 discloses a phthalazine-1 (2) -containing compoundH) The compound has PARP (poly adenosine diphosphate ribose) inhibition effect, so that the compound has antitumor activity, and can achieve the effects of improving the curative effect on tumors and reducing the dosage and toxicity by combining with other antitumor drugs. In the base excision repair pathway, PARP is a key DNA repair enzyme and has the functions of regulating and controlling apoptosis, maintaining genome stability and the like. PARP inhibitionAgents block the DNA repair process by affecting PARP function, causing homologous recombination to repair the missing cells to die. In recent years, PARP has been extensively studied as an anti-tumor target. Olaparib is the first PARP inhibitor on the market and is used for treating recurrent epidermal ovarian cancer, salpingcarcinoma and primary peritoneal cancer. Specifically disclosed in patent CN201810140689.6 are 11 compounds, wherein the structural formula of compound I-9 is as follows:

。

fibrosis (Fibrosis) is self-repair occurring when body tissues are damaged, and can occur in various organs (such as lung, liver, cardiac muscle, kidney, skin and the like), and research shows that in diseases such as chronic obstructive pulmonary disease, pulmonary Fibrosis, liver cirrhosis, systemic sclerosis and the like, all the Fibrosis processes are involved, and the Fibrosis is characterized in that inflammatory reaction, excessive deposition of extracellular matrix and different degrees of Fibrosis of target organs and/or target tissues are all existed. Currently, the targeted treatment of fibrotic diseases has not yet achieved satisfactory results.

Hepatic fibrosis is a pathophysiological process, which refers to abnormal proliferation of connective tissue in the liver caused by various pathogenic factors. Any liver injury has liver fibrosis in the process of liver repair and healing, and if the injury factor cannot be removed for a long time, the fibrosis process can be continuously developed into liver cirrhosis for a long time. There are many causes of hepatic fibrosis, and viral hepatitis, alcoholic liver, fatty liver, autoimmune diseases, etc. are common in clinic. The treatment of anti-hepatic fibrosis mainly comprises: removing pathogenic factors such as antiviral treatment, anti-schistosomiasis treatment, and abstinence from alcohol for hepatitis B and C. The treatment aiming at the hepatic fibrosis is carried out, for example, by inhibiting inflammation or lipid peroxidation, or inhibiting the proliferation and activation of hepatic stellate cells, promoting the degradation of collagen and the like.

Hepatic fibrosis is a common pathological change in chronic liver diseases, and is characterized by increased synthesis and relatively decreased degradation of collagen-based extracellular matrix (ECM), which loses homeostasis, resulting in excessive ECM deposition in the liver to cause hepatic fibrosis. The formation of hepatic fibrosis is caused by the injury, necrosis, apoptosis and liver tissue inflammatory reaction of liver cells caused by various damaged liver factors, and the activation of kupffer cells to secrete various cell factors; the chemical transmitters such as cytokines and lipid peroxidation products secreted by liver cells, platelets, sinus endothelial cells and the like act on hepatic stellate cells together to activate and convert the hepatic stellate cells into myofibroblasts, so that the phenotype and the function of the hepatic stellate cells are changed, the myofibroblasts are proliferated through a paracrine and autocrine mechanism to synthesize a large amount of collagen, proteoglycan and the like, and the generation mechanism is a very complex pathological process and relates to the regulation of histopathology, cytology, cytokines and molecular levels thereof.

It is currently believed that the continuous over-activation of Hepatic Stellate Cells (HSCs) is a key factor in promoting the development of hepatic fibrosis. In normal liver tissue, HSC is usually in a resting state, but when the liver is stimulated by pathological factors such as physical, chemical and microbial infection, HSC is proliferated and activated to be transformed into an activated form, namely Myofibroblasts (MFB), and shows obvious cell proliferation, increased contractility, large expression of alpha-smooth actin (alpha-SMA), and enhanced Collagen synthesis (Collagen I) capacity, and the activated HSC has the following four aspects: (1) conversion to quiescent HSCs; (2) apoptosis of the cell; (3) immune clearance; (4) the cells are aged. The present study shows that HSC is activated and differentiated into MFB, and can be explained by 'three-step cascade reaction', the first step is a prophase inflammatory stage, after hepatic cells are injured by various injury factors, apoptotic or necrotic hepatic cells release a plurality of mitogen-like substances, and HSC is activated to be converted into MFB (direct paracrine pathway); the second step is an inflammatory phase, where liver injury causes inflammatory cells in the liver, mainly activated macrophages and platelets, to secrete large amounts of cytokines such as TGF- β 1, TNF- α, EGF, PDGF, etc., further stimulating the activation of HSCs to convert to MFB (paracrine activation pathway); the third step is the post-inflammatory phase. MFB and HSC can autocrine factors such as TGF-beta 1 and TNF-alpha in transformation, promote self further activation (autocrine activation path), and even remove original liver injury factors in a post-inflammatory stage. It is also sufficient to sustain the hepatic fibrosis formation process, which is an important, possibly perpetuating stage. Therefore, TGF-beta 1 plays an extremely important role in the activation, transformation, differentiation and regulation of HSC and is the strongest HSC fibrosis-promoting factor.

The TGF- β family of signaling pathways plays a key role in regulating different cellular processes, including proliferation, differentiation, migration or cell death, which are critical for the homeostasis of tissues and organs. Migration and proliferation are essential features of activated HSCs, and these are induced at least in part by TGF- β through various mechanisms. Since TGF-. beta.plays an important role in humans, deregulation of its pathways can lead to disease in humans. In the case of the liver, TGF- β signalling is involved in all stages of disease progression, from initial liver injury, to inflammatory reactions and fibrosis, to cirrhosis and cancer. Studies have shown that TGF- β has a key role in inhibiting cell growth and apoptosis in hepatocytes, which is critical for maintaining liver function. TGF-. beta.s are thought to be pro-fibrotic cytokines due to their role in HSC activation and ECM production. The profibrotic effect of TGF-. beta.is primarily mediated by activation of HSCs, and TGF-. beta.is also involved in the development of fibrosis through signaling in other hepatocyte types, such as hepatocytes and hepatic progenitors. TGF-. beta.1 is the most critical cytokine mediating liver injury and fibrosis. Therefore, the search for the compound which can effectively reduce or control the activation of hepatic stellate cells caused by TGF-beta is expected to provide a new medicament for clinically treating hepatic fibrosis.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides the application of the phthalazine derivative in treating hepatic fibrosis diseases, and the invention firstly provides the application of the phthalazine derivative, namely the compound (I), in the field of hepatic fibrosis resistance.

The technical scheme is as follows: in order to achieve the above object, the present invention provides the use of a phthalazine derivative represented by compound (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating hepatic fibrosis diseases:

（I）。

the chemical name of compound (I) is: 4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) -N- (4- ((2-fluorophenyl) carbamoyl) phenyl) piperazine-1-carboxamide.

Wherein the hepatic fibrosis disease is caused by over-activation of hepatic stellate cells.

Wherein, the compound (I) or the pharmaceutically acceptable salt thereof can be used for preparing the medicine for treating the hepatic fibrosis diseases by inhibiting the activation of hepatic stellate cells caused by TGF-beta.

Preferably, the compound (I) is used as a pharmaceutically acceptable salt, which is a salt of the compound with a metal ion or a pharmaceutically acceptable amine or ammonium ion.

Further, the metal ions include sodium, potassium or calcium ions, and the amine includes ethylenediamine or tromethamine.

The invention also provides application of the pharmaceutical composition in preparing a medicament for treating hepatic fibrosis diseases, wherein the pharmaceutical composition comprises the compound (I) or pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier.

Preferably, the composition of the compound and the carrier is in the form of capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalants, ointments, suppositories or patches.

The preparation method and the physical and chemical parameters of the compound (I) are shown in Chinese patent CN 201810140689.6; the synthesis can also be carried out according to the methods of the examples of the present invention.

The invention adopts a Western blot method (refer to basic biochemical experiment guidance, Liuronmei, Lihao Cuo, China agricultural publishing Co.) to detect the expression level of the Collagen I protein in the human semi-activated hepatic stellate cell LX-2. Compared with a control group, if the expression of the Collagen I protein is increased, the activation level of the hepatic stellate cells is increased, and if the expression of the Collagen I protein is decreased, the activation of the hepatic stellate cells is inhibited; and detecting the expression quantity of Collagen I and alpha-SMA proteins in the liver tissues of the mouse model, and comparing with a control group, if the expression quantity of Collagen I and alpha-SMA proteins is increased, indicating that the mouse has hepatic fibrosis, and if the expression quantity of Collagen I and alpha-SMA proteins is decreased, indicating that the hepatic fibrosis degree of the mouse is reduced.

The invention adopts RT-qPCR method (refer to 'molecular biology experimental method and skill', Shunhuangjiang Kabushiki Kaisha's main edition, Ligang Kabushiki Kaisha' and Zhongshan university Press) to detect the Collagen I mRNA content in the semi-activated hepatic stellate cell LX-2 of human, compared with a control group, if the expression of the Collagen I mRNA is increased, the activation level of the hepatic stellate cell is increased, and if the expression of the Collagen I mRNA is decreased, the activation of the hepatic stellate cell is inhibited.

The invention adopts an enzyme-linked immunosorbent assay method (refer to Nanjing constructed ALT/AST kit specification) to detect the levels of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) in mouse serum. Compared with the control group, if the expression of AST and ALT is increased, the liver function of the mice is abnormal, and if the expression of AST and ALT is decreased, the liver function of the mice is improved.

The invention adopts a carbon tetrachloride mouse hepatic fibrosis disease modeling method (refer to ZHao S, ZHANG Z, Yao Z, Shao J, Chen A, ZHANG F, ZHEN S. Tetramethylpyrazine entrapment of pulmonary fibrosis in liver fibrosis viia inhibition of hedgehog signaling in liver fibrosis 2017; 69 (2): 115) 127.) at the end of the experiment, each group of blood and liver samples are collected for subsequent analysis.

The application of the compound (I) can adopt oral administration, injection and other modes in clinical administration.

Generally, the dose range of the compound (I) for human use in the treatment is 1mg to 1000 mg/day. Dosages outside this range may also be used depending on the dosage form and the severity of the disease.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the invention provides the application of the phthalazine derivative compound shown in the compound (I) or the pharmaceutically acceptable salt thereof in preparing the medicine for treating hepatic fibrosis diseases for the first time, in particular the hepatic fibrosis diseases caused by excessive activation of hepatic stellate cells.

According to the pharmacodynamic tests, the compound (I) can inhibit the hepatic stellate cell activation caused by TGF-beta (the protein level and the mRNA level of a single stimulation group Collagen I are reduced averagely by adding the medicines) and has obvious improvement effect on the hepatic fibrosis of a carbon tetrachloride molding mouse, so that the compound (I) has the inhibition effect on the hepatic fibrosis and can be used for treating the hepatic fibrosis. Meanwhile, pharmacodynamic tests for resisting hepatic fibrosis are carried out on a human semi-activated hepatic stellate cell line LX-2 and a carbon tetrachloride molding mouse, and test results further fully prove that the compound (I) has the effect of treating hepatic fibrosis diseases. The phthalazine derivative shown in the compound (I) lays a foundation for developing a medicament with clinical treatment value.

Drawings

FIG. 1 is a diagram showing the experimental results of a hepatic stellate cell line LX-2 Western Blot;

FIG. 2 is a data analysis graph of the experimental result graph of FIG. 1;

FIG. 3 is an analysis diagram of the results of RT-qPCR experiments;

FIG. 4 is a graph showing the results of AST and ALT experiments on mice made of carbon tetrachloride;

FIG. 5 is a chart showing the result of Western Blot experiment for liver tissue of mice modeled by carbon tetrachloride;

FIG. 6 is a data analysis graph of the experimental results graph of FIG. 5;

FIG. 7 is a graph showing the results of the liver tissue section HE, Masson and Sirius Red staining experiments of mice modeled with carbon tetrachloride.

Detailed Description

The present invention is further illustrated by the following examples.

In the examples, the code T-5-9, i.e., Compound (I), is used for the efficacy test.

Example 1

The synthesis method of T-5-9 comprises the following steps:

wherein: boc is tert-butyloxycarbonyl and PyBOP is 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate; DIEA ofN,N-diisopropylethylamine; (Cl)₃CO)₂CO is triphosgene.

4- ((4- ((2-fluorophenyl) carbamoyl) phenyl) carbamoyl) piperazine-1-carboxylic acid tert-butyl ester (2)

4-amino-N- (2-fluorophenyl) benzamide (1) (4.75 g, 20.64 mmol) was dissolved in 30 mL of anhydrous dichloromethane, DIEA (7.19 mL, 41.28 mmol) and a solution of triphosgene (2.04 g, 6.88 mmol) in dichloromethane (20 mL) were added successively at room temperature, and after cooling to 0 ℃ and reacting for 3 hours, the mixture was addedN-Boc-piperazine (5.77 g, 30.96 mmol), stirring overnight at room temperature, TLC detecting the completion of the starting material reaction, filtering, concentrating the filtrate to give a crude product, which is separated by column chromatography (eluent: dichloromethane: methanol =50: 1) to give 7.12g of a yellow solid with 78.0% yield. m.p. 133 ℃ C.

¹H-NMR (300 MHz, DMSO-d₆)δ(ppm): 10.11 (1H, s, CONH), 8.92 (1H, s, CONH), 7.89 (2H, d, J = 8.7 Hz, ArH), 7.60-7.49 (1H, m, ArH), 7.35-7.11 (3H, m, ArH), 6.60 (2H, d, J = 7.5 Hz, ArH), 4.16-4.08 (4H, m, 2CH₂N), 3.52-3.48 (4H, m, 2CH₂N), 1.41 (9H, s, (CH₃)₃C)。

N- (4- ((2-fluorophenyl) carbamoyl) phenyl) piperazine-1-carboxamide hydrochloride (3)

Tert-butyl 4- ((4- ((2-fluorophenyl) carbamoyl) phenyl) carbamoyl) piperazine-1-carboxylate (2) (5.64 g, 12.74 mmol) was dissolved in 100 mL of ethyl acetate, an ethyl acetate solution of saturated hydrogen chloride (10 mL) was added dropwise at room temperature to precipitate a white solid, which was stirred at room temperature for 1 hour, filtered, and the filter cake was washed with ethyl acetate (20 mL. times.3) to give 4.31 g of a white solid in 89.3% yield. m.p. > 250 ℃.

¹H-NMR (300 MHz, DMSO-d₆)δ(ppm): 10.10 (1H, s, CONH), 9.00 (2H, brs, NH, HCl), 8.98 (1H, s, CONH), 7.90 (2H, d, J = 8.8 Hz, ArH), 7.64-7.49 (1H, m, ArH), 7.35-7.11 (3H, m, ArH), 6.60 (2H, d, J = 7.5 Hz, ArH), 4.11-4.00 (4H, m, 2CH₂N), 3.55-3.48 (4H, m, 2CH₂N)。

4- (2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoyl) -N- (4- ((2-fluorophenyl) carbamoyl) phenyl) piperazine-1-carboxamide (T-5-9)

2-fluoro-5- ((4-oxo-3, 4-dihydrophthalazin-1-yl) methyl) benzoic acid (4) (0.75 g, 2.51 mmol) andN- (4- ((2-fluorophenyl) carbamoyl) phenyl) piperazine-1-carboxamide hydrochloride (3) (0.95 g, 2.51 mmol) was dissolved in 10 mLN, NTo dimethylformamide was added PyBOP (1.52 g, 3.01 mmol),N,NDiisopropylethylamine (1.56 mL, 8.93 mmol), stirred at room temperature overnight, TLC detected complete reaction of the starting material, the reaction was poured slowly into water (30 mL), stirred for 1 hour to precipitate a solid, filtered under suction, and the filter cake was washed with water (5 mL. times.2) to give the crude product. The crude product was isolated by column chromatography (eluent: dichloromethane: methanol =50: 1) to give 1.3 g of a white solid with a yield of 83.2%.

¹H-NMR(300MHz, DMSO-d₆)δ(ppm): 12.63 (s, 1H, CONHN), 9.93 (s, 1H, CONH), 8.96 (s, 1H, CONH), 8.30 (d, J = 8.5 Hz, 1H, ArH), 8.01-7.83 (m, 5H, ArH), 7.63 (d, J = 9.2 Hz, 2H, ArH), 7.47-7.41 (m, 1H, ArH), 7.36-7.33 (m, 1H, ArH), 7.33-7.21 (m, 5H, ArH), 4.36 (s, 2H, ArCH₂), 3.73-3.68 (m, 2H, CH₂N), 3.62-3.57 (m, 2H, CH₂N), 3.46-3.42 (m, 2H, CH₂N), 3.29-3.24 (m, 2H, CH₂N). HRMS (EI): m/z [M+1]⁺ calcd for C₃₄H₂₉F₂N₆O₄: 623.2218, found 623.2222。

Example 2

Human semi-activated hepatic stellate cell line LX-2 cell assay

The LX-2 cell culture mode of this example: the LX-2 cells were removed from the freezer at-80 ℃ and cultured in DMEM medium containing 10% fetal bovine serum at 10 cm²In a culture dish, 5% CO at 37 ℃₂Culturing in an incubator, changing the culture solution, carrying out passage, and taking 3-6 generations of cells for experiment.

Western Blot experiment

6-well plates were seeded with human semi-activated hepatic stellate cells LX-2 (5X 10)⁵One/well), a blank control group, a TGF-beta stimulation group, a T-5-9 (0.1 mu M) group, a T-5-9 (1 mu M) group and a fifth T-5-9 (10 mu M) group are established. When the cell density in the hole is 70%, treating the cells in the mode shown in the table 1, quantitatively collecting the lysed protein after 24 h, detecting the expression amount of the Collagen I protein in the LX-2 by using a Western blot method, analyzing the result, and comparing the expression conditions of the Collagen I in a TGF-beta stimulation group and a drug adding group to test whether the T-5-9 can inhibit the LX-2 activation caused by the TGF-beta from the protein level.

TABLE 1 cell treatment protocol for each test group

The experimental results are shown in FIG. 1, and FIG. 1 shows the contents of the target protein Collagen I and the reference protein GAPDH in the cell protein sample, and the color shades represent the contents.

As can be seen from fig. 1:

the expression levels of Collagen I and GAPDH in LX-2 cells are shown as blank control group.

Secondly, the expression level of Collagen I and GAPDH in cells after LX-2 cells are stimulated and activated by adding TGF-beta is shown by representing a TGF-beta stimulation group, and the expression level of Collagen I and GAPDH in cells is obviously enhanced compared with that of the first group.

③ represents the T-5-9 (0.1. mu.M) group, and shows that the expression levels of Collagen I and GAPDH in cells after the LX-2 cells are stimulated by adding TGF-beta and administered with T-5-9, are reduced compared with the Collagen I in the group (C).

And fourthly, representing the T-5-9 (1 mu M) group, showing that the expression levels of Collagen I and GAPDH in cells after the LX-2 cells are stimulated by adding TGF-beta and are simultaneously administrated by using the T-5-9, and the expression of the Collagen I is reduced and is restored to the background level of the group I compared with the group II.

Fifthly, represents the group T-5-9 (10 mu M), and shows that the expression levels of Collagen I and GAPDH in cells after the LX-2 cells are stimulated by adding TGF-beta and are simultaneously administrated by using T-5-9, compared with the group II, the expression of Collagen I is obviously reduced and is lower than the background level of the group I.

The analysis of the test results is shown in FIG. 2. Importing the graph 1 into Image processing software Image J to perform gray level analysis to obtain a gray level value of each protein band, dividing a target protein Collagen I gray level value in each group by a GAPDH protein gray level value to obtain a target protein Collagen I relative gray level value, importing the target protein Collagen I relative gray level value into Graphpad software to make a histogram, and obtaining the graph 2. (refer to Lujin, east of Wedng, Yangyongqing, etc.. influence of different parameters on Western blotting results. Biotechnology, 29 (4): 371 Aug.2019).

As can be seen from fig. 2: compared with the blank control group, the TGF-beta stimulated group Collagen I is increased; compared with the TGF-beta stimulation group, Collagen I in the Tc-5-9 administration group is reduced and shows dose dependence.

As can be seen from fig. 1 and 2: t-5-9 (i.e., Compound (I)) was able to dose-dependently inhibit hepatic stellate cell activation by TGF-. beta. (Collagen I elevation). Therefore, T-5-9 has the efficacy of treating hepatic fibrosis.

II, QPCR experiments

6-well plates were seeded with human semi-activated hepatic stellate cells LX-2 (5X 10)⁵One/well), a blank control group, a TGF-beta stimulation group and a T-5-9 group are established. When the cell density in the pores is 60%, the cells are treated in the mode shown in the table 2, TRIZOL is added after 24 hours to extract cell RNA and invert the cell RNA into DNA, the content of the Collagen I mRNA in the LX-2 is detected by adopting an RT-qPCR method, and whether the T-5-9 can inhibit the activation of the LX-2 caused by TGF-beta from the mRNA level (the expression of the Collagen I mRNA is enhanced) is analyzed.

TABLE 2 treatment of each group of cells

Analysis of experimental results As shown in FIG. 3, the height of the column in FIG. 3 indicates the relative level of the gene of interest (Collagen I) mRNA in the cellular mRNA sample. (refer to the application of Guanzhu, Hauyita, Guoshi. GraphPad Prism software in the auditing of medical paper data. technology dissemination, 1674-.

As can be seen from fig. 3: compared with a blank control group, the expression of the Collagen I mRNA of the TGF-beta stimulation group is obviously enhanced; compared with the TGF-beta stimulation group, the Collagen I mRNA expression of the T-5-9 group stimulated by the TGF-beta and added with the T-5-9 is remarkably reduced, thereby indicating that the T-5-9 can inhibit the hepatic stellate cell activation (the Collagen I mRNA expression is enhanced) caused by the TGF-beta and has the inhibiting effect on hepatic fibrosis.

Example 3

Anti-hepatic fibrosis animal test

The model building method of the mouse hepatic fibrosis disease model comprises the following steps: 24 male mice (6 weeks old, 18-22 g in weight) were randomly divided into 4 groups of 6 mice each. By injection of 10% carbon tetrachloride (CCl)₄0.5 ml/100 g body weight) for 8 weeks (three times per week) to induce liver fibrosis in wild-type mice. Injecting the same amount of olive oil into the abdominal cavity of the control group mice to replace carbon tetrachloride; injecting carbon tetrachloride into the abdominal cavity of the model group mice; positive medicine group mouse abdominal cavity injection CCl₄And colchicine (0.1 mg/kg, once daily, weeks 5-8); treatment group mice intraperitoneal injection CCl₄And T-5-9 (12.5 mg/kg, once daily, week 5-8); the subsequent experiments and figures were designated as: the medicine comprises a control group, a model group, a positive medicine group and a T-5-9 group. At the end of the experiment, groups of blood and liver samples were collected for subsequent analysis.

First, mouse serum AST, ALT experiment

Taking mouse serum samples, and detecting the AST and ALT contents in the mouse serum according to the detection method of Nanjing constructed AST and ALT kit by 3 samples in each group.

The analysis of the test results is shown in FIG. 4, and the heights of the left and right columns in FIG. 4 show the AST and ALT contents in the mouse serum respectively.

As can be seen from FIG. 4, compared with the control group, the AST and ALT contents of the model group are obviously increased, and the liver function of the mouse is abnormal; comparing the model group, the AST and ALT contents of the positive medicines are obviously reduced, which shows that the liver function of the mouse is recovered after the positive medicine colchicine treatment; compared with the model group, the AST and ALT contents of the T-5-9 groups are obviously reduced, which shows that the liver function of the mice is recovered after the T-5-9 treatment. Therefore, the T-5-9 can restore the liver function abnormal index in the mouse liver fibrosis model and has a relieving effect on liver fibrosis.

Second, mouse liver tissue experiment

Taking the mouse liver tissue sample, extracting protein by RIPA strong lysate, then making the sample quantitatively, detecting the expression quantity of Collagen I and alpha-SMA protein in the liver tissue by Western blot method, analyzing the result, and judging whether T-5-9 can play the role of anti-hepatic fibrosis on the animal model by comparing the expression conditions of the Collagen I and alpha-SMA protein of the model group and the drug adding groups (the positive drug group and the T-5-9 group).

The analysis of the test results is shown in FIG. 5, and FIG. 5 shows the content of the target proteins Collagen I, alpha-SMA and the internal reference protein GAPDH in the cell protein sample, and the shade of color represents the content. As can be seen from fig. 5:

compared with the control group, the expression of the model group Collagen I and alpha-SMA is increased, which indicates that the mouse has hepatic fibrosis and the model is effective.

Compared with the model group, the expression of the positive medicines Collagen I and alpha-SMA is reduced, which shows that the positive medicine colchicine has the treatment effect on the hepatic fibrosis of the mice.

Compared with the model group, the expression of the Collagen I and the alpha-SMA in the T-5-9 group is reduced, which shows that the T-5-9 group has the treatment effect on the hepatic fibrosis of the mice.

The analysis of the test results is shown in FIG. 6. Importing the graph 5 into Image processing software Image J to perform gray level analysis to obtain a gray level value of each protein band, dividing the gray level values of the target proteins Collagen I and alpha-SMA in each group by the gray level value of the GAPDH protein to obtain the relative gray level values of the target proteins Collagen I and alpha-SMA, importing the relative gray level values into Graphpad software to prepare a histogram to obtain a graph 6.

As can be seen from fig. 6: CCL was used in control group and model group mice₄The expression of the modeling Collagen I and the alpha-SMA is increased, and hepatic fibrosis is generated; CCL was used to compare model groups, positive drug groups and T-5-9 groups of mice₄After the model is made, positive colchicine and T-5-9 are respectively used for treatment, the expression of Collagen I and alpha-SMA is reduced, and the hepatic fibrosis is inhibited.

As can be seen from fig. 5 and 6: t-5-9 has therapeutic effect on hepatic fibrosis mouse.

Third, mouse liver tissue section staining experiment

Freshly harvested mouse liver tissue was fixed in 4% paraformaldehyde overnight at room temperature. After tissue fixation was complete, embedding, sectioning and HE, Sirius Red and Masson staining were performed.

The results of the experiments are shown in FIG. 7, and FIG. 7 shows the staining of liver tissue sections by HE, Masson and Sirius Red. As can be seen from fig. 7:

control HE, Masson and Sirius Red staining all showed normal morphology.

Comparing with a control group, and displaying degeneration and necrosis of liver cells by HE staining of a model group, wherein a large-area false lobular tissue structure is presented; masson staining showed a large amount of blue collagen fiber deposits extending outward from around the zone of confluence, with thicker fiber cords and darker staining, indicating more collagen fibers; sirius Red staining showed massive Red type I collagen fiber deposition, thus indicating that mice develop liver fibrosis and the model is effective.

Comparing the model group, positive drug HE, Masson and Sirius Red stain towards normal shape, which shows that the positive drug colchicine has therapeutic effect on hepatic fibrosis of mice.

Comparing the model group, the T-5-9 group staining by HE, Masson and Sirius Red tended to normal shape, which indicates that T-5-9 has therapeutic effect on hepatic fibrosis of mice.

The test proves that T-5-9, namely the compound (I), can inhibit the activation of hepatic stellate cells and treat the hepatic fibrosis of mice and has the treatment effect on hepatic fibrosis diseases.

Claims

1. The use of a phthalazine derivative represented by compound (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating hepatic fibrosis caused by overactivation of hepatic stellate cells:

。

2. the use according to claim 1, wherein the compound (I) or a pharmaceutically acceptable salt thereof is used for the manufacture of a medicament for treating liver fibrosis caused by excessive activation of hepatic stellate cells by inhibiting activation of hepatic stellate cells by TGF- β.

3. Use of a pharmaceutical composition for the manufacture of a medicament for the treatment of liver fibrosis caused by overactivation of hepatic stellate cells, wherein said pharmaceutical composition comprises compound (I) according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier.

4. The use according to claim 3, wherein the composition of the compound and the carrier is in the form of capsules, powders, tablets, granules, pills, injections, oral liquids, inhalants, ointments, suppositories, or patches.